Hypercalcemia in malignancy appears to result most commonly from accelerated bone resorption induced by soluble factors secreted by tumor cells. The identity of these factors and their mechanism(s) of action, however, are largely unknown. We have identified a potent bone-resorbing factor in medium of cultured tumor cells from a patient with transitional-cell carcinoma ("TCC") of the bladder, hypercalcemia, and no bone metastases. These cells are tumorigenic and cause hypercalcemia when implanted in athymic nude mice. A bone-resorbing factor identical to that in TCC-cell culture medium was found in the patient's urine and in extracts of tumors grown in nude mice. This factor, a peptide of approximately 13,000 daltons, survives denaturation (8M urea), reduction (65mM DTT), and heating (56~C) and causes bone resorption by a mechanism dependent upon stimulation of bone prostaglandin synthesis. This factor is different from others recently described as it does not stimulate adenylate cyclase and lacks biologic and chemical features of transforming growth factors. The major goals of this proposal are to purify this factor in quantities sufficient to determine its structure, to develop specific antibodies directed against it, and to further test its biologic actions in vivo and in vitro. We will purify this factor to homogeneity from conditioned medium of TCC cell cultures on the basis of its bone-resorbing activity in a sensitive in vitro bioassay. Structure of the purified peptide will be determined using standard chemical techniques, complemented by microsequence analysis of labeled factor prepared by biosynthetic incorporation of radioactive amino acids. Monoclonal antibodies to the factor will be prepared by in vitro or in vivo immunization of mice and used to develop immunoaffinity reagents for factor purification and radioassays for its measurement. Sufficient purified factor will be obtained to permit direct study of its biologic actions in vivo and in vitro and, thereby, to enable us to test the hypothesis that this tumor-derived bone-resorbing factor is the cause of the hypercalcemia induced by this tumor in vivo. These studies may have profound implications for understanding normal bone development and remodeling as well as the pathophysiology of the humoral hypercalcemia of malignancy. (C)